Mdck-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells

ABSTRACT

Disclosed is a Madin-Darby canine kidney (MDCK)-derived cell line. The MDCK-derived cell line is derived from MDCK cells deposited under accession number ATCC CCL-34. The MDCK-derived cell line can be prepared by serum-free culture and suspension culture. Preferably, the MDCK-derived cell line has low or no tumorigenicity. The MDCK-derived cell line is preferably selected from MDCK Sky1023, MDCK Sky10234 and MDCK Sky3851. Further disclosed are a culture method for growing the MDCK-derived cells and a method for producing a vaccine virus using the MDCK-derived cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/KR2011/006041 filed on Sep. 6, 2011, which claims priority to KoreanPatent Application No. 10-2011-0085902 filed on Aug. 26, 2011, whichapplications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a novel MDCK-derived cell line that canbe prepared by serum-free culture and suspension culture without theneed to be attached to carriers, a method for preparing the MDCK-derivedcell line, and a method for producing a vaccine virus using theMDCK-derived cell line.

BACKGROUND ART

Fertilized eggs, mouse brains, primary cells and established cells aregenerally used as sources for the production of vaccines. However, suchtraditional vaccine sources have several problems. For example, when itis intended to use fertilized chicken eggs for vaccine production, thereare difficulties in raising chickens, managing the fertilized eggsdepending on vaccine production schedules and purifying ingredientsderived from egg proteins.

Fetal calf serum is generally added as a growth factor for cell culture.However, serum is susceptible to contamination with prions and virusesand its commercial products might vary in quality. Moreover,high-quality fetal calf serum products derived from Australian and NewZealand calves are highly priced, resulting in an increase in productioncost.

MDCK cell lines are established cell lines where various kinds ofviruses can proliferate. Since MDCK cell lines exhibit a strong tendencyto attach to other surfaces, large-area culture vessels and carriers arerequired for large-scale culture, incurring considerable costs.Specifically, investment costs for culture equipment or carriers arevast and a processing step is needed to remove cells attached tocarriers, posing the risk of loss of and damage to the cells. Thus,there is a need for a cell line that can be prepared by serum-freeculture and suspension culture and is thus suitable for use in theproduction of a vaccine through animal cell culture in an economical andsafe manner.

U.S. Pat. No. 6,825,036 discloses an MDCK-derived cell line that can begrown in serum-free culture and in suspension culture without a solidcarrier. The U.S. patent employs two approaches for the adaptation ofthe cell line to suspension culture. According to the first approach,the MDCK-derived cell line is obtained by direct suspension culture in aspinner flask. In this case, the cell density does not reach 1.0×10⁶cells/ml, a level necessary for industrial-scale production. Accordingto the second approach, the MDCK-derived cell line is obtained byculturing the original MDCK cells in the presence of beads as carriers,expanding the culture scale and growing the cultured cells in theabsence of carriers. The second approach has the disadvantage in thatthe procedure is relatively complicated.

There are several reports that original MDCK cell lines are tumorigenic.Thus, there exists a danger of potential tumorigenicity when originalMDCK cell lines are used for vaccine production. Under thesecircumstances, there is a need to develop a novel cell line that can beprepared by serum-free culture and suspension culture and preferably hasvery low tumorigenicity or is non-tumorigenic.

SUMMARY

The present invention is designed to solve the problems of the priorart, and therefore it is an object of the present invention to providean MDCK-derived cell line that can be advantageously prepared byserum-free culture and suspension culture and has very low or notumorigenicity as compared to original MDCK cell lines that can be usedto grow vaccine viruses. It is another object of the present inventionto provide a method for producing a virus, particularly an influenzavirus, using the cell line.

In order to achieve the above objects, the present invention provides aparticular Madin-Darby canine kidney (MDCK)-derived cell line that isderived from MDCK cells deposited under accession number ATCC CCL-34,does not require serum for cell growth and can be prepared by suspensionculture without the need to be attached to carriers.

The MDCK-derived cell line of the present invention can be preparedthrough the following steps: S1) adapting an original MDCK cell line toa serum-free medium to prepare a cell line adapted to the serum-freeculture; and S2) directly adapting the cell line adapted to theserum-free culture to suspension culture without undergoing carrieradaptation and screening the desired cell line.

More specifically, the MDCK-derived cell line of the present inventioncan be prepared by a method including (a) preparing original MDCK cellsdeposited under accession number ATCC CCL-34, (b) adapting the originalMDCK cells to a chemically defined serum-free medium to allow theoriginal MDCK cells to grow in the serum-free medium, and (c) inducingdirect adaptation of the adherent MDCK cells adapted in step (b) tosuspension culture in a chemically defined serum-free medium withoutundergoing carrier adaptation to allow the MDCK cells to grow in asuspension state without carriers.

The MDCK-derived cell line newly established by the method can beprepared by serum-free culture and suspension culture. The MDCK-derivedcell line of the present invention is preferably selected from MDCKSky1023 (DSM ACC3112), MDCK Sky10234 (DSM ACC3114) and MDCK Sky3851 (DSMACC3113), which have been deposited at the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ), Braunschweig, Germany, on Jan.27, 2011. The term “serum-free medium” as used herein means a medium towhich serum is not substantially added and in which the established cellline of the present invention can be prepared by culture.

The expression “serum is not substantially added” means that serum ispresent in an amount of 0.5 vol % less, preferably 0.1 vol % or less,more preferably 0.01 vol % or less, most preferably not present at all.

U.S. Pat. No. 6,825,036 employs two approaches to prepare anMDCK-derived cell line in a serum-free medium by suspension culture. Thefirst approach tries direct suspension culture of an original MDCK cellline in a spinner flask. In this case, the cell density does not reach1.0×10⁶ cells/ml, a level necessary for industrial-scale production.According to the second approach, the MDCK-derived cell line is obtainedby culturing the original MDCK cells in the presence of carriers andgrowing the cultured cells in the absence of carriers.

In contrast, the present invention tries direct culture of original MDCKcells in a spinner flask without undergoing carrier adaptation toprepare a newly established cell line that can be prepared in aserum-free medium by suspension culture. The established cell line B-702of U.S. Pat. No. 6,825,036 reaches a cell density of 1.0×10⁶ cells/mlwithin a week, whereas the established cell line of the presentinvention reaches a cell density of at least 1.0×10⁶ cells/ml withinabout 3 days, indicating much superior proliferation potential.

Several reports state that previously known MDCK cell lines aretumorigenic. In contrast, the established cell line of the presentinvention has very low or no tumorigenicity when compared to the knownMDCK cell lines. Specifically, the newly established MDCK-derived celllines prepared in the Examples section were confirmed to have very lowor no tumorigenicity when compared to existing MDCK cell lines throughtests using the cell lines, cell lysates and cell DNAs in nude mice.From these test results, it can be concluded that the MDCK-derived cellline of the present invention is stable enough to be used for vaccineproduction. More preferably the MDCK-derived cell line of the presentinvention can be prepared by serum-free culture and suspension culture.

The present invention also provides a method for producing a vaccinevirus using the MDCK-derived cell line. Examples of viruses that can begrown using the MDCK-derived cell line include influenza viruses,measles viruses, Japanese encephalitis viruses, mumps viruses, rubellaviruses, polio viruses, HSV-1, HSV-2, rabies viruses, RS viruses,reovirus type 3, yellow fever virus, parvoviruses, coxsackie viruses,adenovirus types 1 to 47, Lassa viruses and vacciniaviruses. TheMDCK-derived cell line of the present invention is most suitable for usein the production of influenza viruses.

More specifically, the present invention provides a method for producingan influenza virus from a cell culture, the method including: (a)inoculating a serum-free culture medium with the MDCK-derived cells at aconcentration of about 1×10⁴ to about 1×10⁶ cells/ml; (b) allowing theMDCK-derived cells to grow in a disposable bioreactor system until thecell density reaches at least about 5×10⁶ cells/ml, including culturingthe MDCK-derived cells while maintaining one or more culture conditionsselected from the group consisting of a stirring rate of about 40 toabout 100 rpm, a pH of about 6.5 to about 7.5 and a dissolved oxygen(DO) concentration of about 35 to about 100%; (c) infecting the grownMDCK-derived cells with an influenza virus; (d) culturing the infectedgrown MDCK-derived cells under conditions allowing cloning of theinfluenza virus; and (e) isolating the influenza virus from the cellculture composition. Preferably, the influenza virus production methodof the present invention further includes adding a fresh medium to thecell culture or replacing a portion of the medium with a fresh medium instep (b).

The present invention also provides a virus or a virus antigen producedby the virus production method. The present invention also provides animmunogenic pharmaceutical composition including the virus antigen.

The novel MDCK-derived cell line of the present invention can beprepared by serum-free culture and suspension culture and has theadvantage of very low or no tumorigenicity. In addition, the novelMDCK-derived cell line of the present invention can be efficiently usedfor virus proliferation. Furthermore, the novel MDCK-derived cell lineof the present invention is suitable for use in the production of avirus, particularly an influenza virus.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings illustrate preferred embodiments of theinvention and, together with the foregoing disclosure, serve to providefurther understanding of the technical spirit of the invention. However,the present invention is not to be construed as being limited to thedrawings.

FIG. 1 shows cell growth profiles of MDCK-derived cell lines duringsuspension culture in spinner flasks;

FIG. 2 is a flow chart showing a representative purification process ofan influenza virus; and

FIG. 3 shows HI serum test results obtained in animal experiments usingpurified samples of viruses grown in MDCK-derived cell lines.

DETAILED DESCRIPTION

The following examples are provided to assist in a further understandingof the invention. However, these examples may take several other forms,and the scope of the invention should not be construed as being limitedthereto. These examples are provided to more fully explain the inventionto those having ordinary knowledge in the art to which the inventionbelongs.

Example 1 Preparation of MDCK-Derived Cell Lines by Serum-Free Cultureand Suspension Culture

CCL-34, an MDCK cell line, was furnished from ATCC. CCL-34 was culturedin an EMEM medium supplemented with 10% serum in a T-25 flask at 37° C.and 5% CO₂. After the cells were expanded, they were cultured in amedium consisting of the EMEM medium and a serum-free medium (50%). Itwas confirmed whether growth of the cells was normal or not duringculture. When growth of the cells was confirmed to be normal, the growncells were cultured in a medium containing a serum-free medium (75%).This procedure was repeated to obtain a cell line adapted to aserum-free medium (100%). EX-CELL MDCK (Sigma), UltraMDCK (Lonza) andVP-SFM (Invitrogen) may be used as the media for serum-free culture.

The cell line adapted to the serum-free medium was sufficiently expandedin a T-flask. Thereafter, the expanded cell line was adapted tosuspension culture with stirring at a rate of 40-80 rpm in a spinnerflask (Corning) at 37° C. and 5% CO₂. When the pH of the culture mediumwas decreased or the cells were grown above a predetermined level, themedium was exchanged with a new one or the cells were subcultured. Afteradaptation to suspension culture for 3-6 months, the cell concentrationreached 2×10⁶ cells/ml or more. The cell viability was 95% or above andsuspension culture into single cells was observed. EX-CELL 302 CHO(Sigma), UltraCHO (Lonza) and SMIF-6 (Invitrogen) may be used as themedia for suspension culture. As a result of the serum-free culture andsuspension culture, three kinds of MDCK-derived cell lines were obtainedand termed MDCK Sky1023, MDCK Sky10234 and MDCK Sky3851.

Example 2 Evaluation of Proliferation Potentials of the Cell Lines

The MDCK-derived cell lines prepared by culture in the serum-free mediawere cultured under the conditions indicated in Table 1. Theproliferation potentials of the MDCK-derived cell lines were evaluated.The MDCK cell line (ATCC CCL-34) as a control was grown in a mediumsupplemented with 10% serum.

TABLE 1 MDCK-derived cell lines Control (ATCC CCL-34) Culture EX-CELLMDCK (Sigma), EMEM (Lonza) media UltraMDCK (Lonza), VP-SFM (Invitrogen)Additives L-Glutamine (Lonza) L-Glutamine (Lonza) 2% v/v, 2% v/v Fetalcalf serum (Lonza) 10% v/v Culture 37° C., 5% CO₂, moist 37° C., 5% CO₂,moist conditions Culture T-75 flask (15 ml) T-75 flask (15 ml) volume

The cell concentrations were about 1.0×10⁵ cells/ml at the beginning ofculture. When the cell concentrations reached about 1×10⁶ cells/ml or3-4 days after the culture, the cells were subcultured. The cellconcentrations at the beginning of subculture were adjusted to 1×10⁵cells/ml.

Each of the MDCK-derived cell lines grown in the serum-free media showeda cell growth rate comparable to that of the MDCK cell line grown in theserum medium.

Example 3 Evaluation of Proliferation Profiles and Subculture Stabilityof the Cell Lines

After the three kinds of cell lines adapted to serum-free culture andsuspension culture were continuously cultured in respective spinnerflasks, their proliferation profiles and subculture stability wereevaluated. The cell concentrations at the beginning of culture wereadjusted to about 4×10⁵ cells/ml. About 3-4 days after the culture, thecell concentrations reached about 2×10⁶ cells/ml or more. The culturewas conducted under the following conditions. The results are shown inFIG. 1.

Initial cell concentration: 4×10⁵ cells/ml

Culture scale: 50 ml spinner flask

Spinner rotational rate: 60 rpm

Culture conditions: 37° C., 5% CO₂, moist

Subculture condition: 3-4 days after culture

Example 4 Evaluation of Virus Proliferation

2010/2011 seasonal influenza vaccine strains were grown using theMDCK-derived cells under the following suspension culture conditions:

Cell concentration: 2×10⁶ cells/ml

Culture scale: 1,000 ml spinner flask

Spinner rotational speed: 60 rpm

Culture conditions: 34° C., 5% CO₂, moist

Culture period: 3 days

It is commonly known that influenza viruses are better grown duringculture at 34° C. than at 37° C., which was experimentally confirmed.Trypsin was included in culture media to infect the cell lines with theinfluenza viruses during culture. The titers of the influenza viruseswere measured by haemagglutination assay. After culture for 3 days, mostof the cells were killed. The culture supernatants were collected andthe titers of the viruses were measured. The results are shown in Table2. As can be seen from the results in Table 2, most of the virusesshowed HA titers of 1024 or more. The growth levels of the viruses weresimilar to those of eggs or MDCK cells cultured in 10% FBS containingmedia. These results have proved that the MDCK-derived cell lines aresuitable for efficient production of viruses under serum-free cultureand suspension culture conditions.

TABLE 2 Virus MDCK Sky1023 MDCK Sky10234 MDCK Sky3851A/California/07/2009 (H1N1) 512 1024 2048 A/perth/16/2009 (H3N2) 10241024 4096 B/Brisbane/60/2008 4096 2048 4096

Example 5 Identification of the Ability of the Grown Viruses to FormAntibodies

To identify the ability of the influenza viruses grown in theMDCK-derived cells to form antibodies, the viruses were purified fromthe culture solutions and inoculated into mice. The antibody values ofthe viruses were measured. A flow chart of the purification process isshown in FIG. 2, and a brief explanation thereof is given below.

First, each of the virus culture solutions was centrifuged to removecell lysates therefrom and filtered through a 0.45 μm filter. Thefiltrate was concentrated by ultrafiltration using a 300 kDa cut-offcartridge, and the virus was inactivated with formalin. Thereafter, thevirus was isolated from the culture solution by ultracentrifugationthrough a sucrose concentration gradient. The virus was disrupted bytreatment with Triton X-100, concentrated by ultrafiltration to removethe detergent, filtered through a 0.2 μm filter to sterilize, affordinga vaccine solution.

The purified solutions of the virus strain A/California/07/2009 (H1N1)grown using the three MDCK-derived cell lines MDCK Sky1023, MDCKSky10234 and MDCK Sky3851 were used for animal experiments to identifythe efficacy of the virus strain. As a control, a 08/09 seasonalinfluenza vaccine (IVR-148, NYMC X-175C, B/Florida/4/2006) was used. Atotal of six test groups were used, each of which was inoculated intofive mice. Serum samples from the five mice were collected. Ahaemagglutination inhibition (HI) test was conducted on the collectedserum to measure the antibody value of the virus.

Serum was obtained by drawing blood from the retro-orbital plexus of themice using a capillary tube prior to injection (0 week) of the testgroup into the mice. After the blood drawing, a total of 200 μl of thetest group was injected into the hind legs (100 μl for each leg) of eachof the mice via the IM route. Two weeks after the injection, serum wasobtained by drawing blood from the retro-orbital plexus in the samemanner as above. The same amount of the test group as that injected at 0week was inoculated into each of the mice to boost the ability to formantibodies. Four weeks after the injection, serum was obtained bydrawing blood from the retro-orbital plexus in the same manner as above.

The obtained serum samples were treated by the following procedure. Theserum samples drawn from the mice were collected and divided into 30 μlsamples. 90 μl of RDE was added to each 30 μl sample and was allowed tostand at 37° C. for at least 18 hr. The mixture was further leftstanding at 56° C. for at least 30 min to inactivate the RDE.Subsequently, 120 μl of 0.85% physiological saline and 15 μl (a volumecorresponding to 1/20 of the total volume) of chicken red blood cellswere subsequently added, sufficiently suspended, and left standing at 4°C. for 1 hr. The settled blood cells were re-suspended at 30-minintervals. Thereafter, the suspension was centrifuged at 1,200 rpm for10 min to separate the serum. HI test was conducted on the serum tomeasure the antibody value of the serum. The measured results are shownin Table 3. All experimental groups were measured to have HI titers of40 or more. That is, when the virus cultured from the MDCK-derived cellswas injected into the animals, similarly to the existing vaccine, it wasconfirmed that antibodies against the virus were formed. From theseresults, it can be concluded that the MDCK-derived cell lines are usefulfor the production of virus vaccines.

TABLE 3 Experimental Amount of antigen group Cell line inoculated HItiter 1 MDCK Sky1023  15 μg/mouse 80 2  MDCK Sky10234  15 μg/mouse 160 3 MDCK Sky10234 7.5 μg/mouse 160 4 MDCK Sky3851  15 μg/mouse 80 5 MDCKSky3851 7.5 μg/mouse 80 6 Reference vaccine 320

Example 6 Identification of Tumorigenicity of the Cell Lines in NudeMice

To evaluate the tumorigenicity of the cell lines MDCK Sky1203 and MDCKSky3851, the MDCK-derived cell lines and substances derived therefrom(including cell lysates and cell DNAs) were transplanted into thehypodermis of BALB/c-nu/nu mice as test animals, as indicated in Table4. An observation was made to determine whether tumors were formed for12 weeks. The cells were inoculated in doses of 10¹, 10³, 10⁵ and 10⁷cells, the cell lysates were inoculated in doses of 10⁵ and 10⁷ cells,and the cell DNAs were inoculated in doses of 10⁵ and 10⁷ cells. Each ofthe groups was inoculated into 5-10 mice. The experimental results areshown in Table 4. The cell lines MDCK Sky1023 and MDCK Sky3851 wereconfirmed to have low or no tumorigenicity when compared to the originalMDCK (ATCC) cell line as an internal control.

TABLE 4 Experimental Experimental Number of animals where group sampleDose tumor was found Negative control PBS 0/5 Positive controls HeLaCells 10⁵ 1/5 HeLa Cells 10⁷ 5/5 Internal groups MDCK 10¹  0/10 MDCK 10³ 0/10 MDCK 10⁵ 10/10 MDCK 10⁷  9/10 MDCK Sky1023 Cells 10¹  0/10 10³ 0/10 10⁵  3/10 10⁷ 10/10 Cell lysates 10⁵ 0/5 10⁷ 0/5 DNAs 10⁵ 0/5 10⁷0/5 MDCK Sky3851 Cells 10¹  0/10 10³  0/10 10⁵  0/10 10⁷  0/10 Celllysates 10⁵ 0/5 10⁷ 0/5 DNAs 10⁵ 0/5 10⁷ 0/5

What is claimed is:
 1. A Madin-Darby canine kidney (MDCK)-derived cellline that is derived from MDCK cells deposited under accession numberATCC CCL-34, does not require serum for cell growth and is prepared bysuspension culture without the need to be attached to carriers.
 2. Amethod for producing a vaccine virus using the MDCK-derived cell lineaccording to claim
 1. 3. The method according to claim 2, wherein thevirus is selected from the group consisting of influenza viruses,measles viruses, Japanese encephalitis viruses, mumps viruses, rubellaviruses, polio viruses, HSV-1, HSV-2, rabies viruses, RS viruses,reovirus type 3, yellow fever virus, parvoviruses, coxsackie viruses,adenovirus types 1 to 47, Lassa viruses and vacciniaviruses.
 4. Themethod according to claim 3, wherein the virus is an influenza virus. 5.The MDCK-derived cell line according to claim 1, wherein theMDCK-derived cell line has low or no tumorigenicity as compared to theoriginal MDCK cell line.
 6. A method for producing a vaccine virus usingthe MDCK-derived cell line according to claim
 5. 7. The method accordingto claim 6, wherein the virus is selected from the group consisting ofinfluenza viruses, measles viruses, Japanese encephalitis viruses, mumpsviruses, rubella viruses, polio viruses, HSV-1, HSV-2, rabies viruses,RS viruses, reovirus type 3, yellow fever virus, parvoviruses, coxsackieviruses, adenovirus types 1 to 47, Lassa viruses and vacciniaviruses. 8.The method according to claim 7, wherein the virus is an influenzavirus.
 9. The MDCK-derived cell line according to claim 1, wherein theMDCK-derived cell line is MDCK Sky1023 (DSM ACC3112), MDCK Sky10234 (DSMACC3114) or MDCK Sky3851 (DSM ACC3113).
 10. A method for producing avaccine virus using the MDCK-derived cell line according to claim
 9. 11.The method according to claim 10, wherein the virus is selected from thegroup consisting of influenza viruses, measles viruses, Japaneseencephalitis viruses, mumps viruses, rubella viruses, polio viruses,HSV-1, HSV-2, rabies viruses, RS viruses, reovirus type 3, yellow fevervirus, parvoviruses, coxsackie viruses, adenovirus types 1 to 47, Lassaviruses and vacciniaviruses.
 12. The method according to claim 11,wherein the virus is an influenza virus.
 13. A method for producing aninfluenza virus from a cell culture, the method comprising: (a)inoculating a serum-free culture medium with the MDCK-derived cellaccording to claim 1 at a concentration of 1×10⁴ to 1×10⁶ cells/ml; (b)allowing the MDCK-derived cells to grow in a disposable bioreactorsystem until the cell density reaches at least 5×10⁶ cells/ml,comprising culturing the MDCK-derived cells while maintaining one ormore culture conditions selected from the group consisting of a stirringrate of 40 to 100 rpm, a pH of 6.5 to 7.5 and a dissolved oxygen (DO)concentration of 35 to 100%; (c) infecting the grown MDCK-derived cellswith an influenza virus; (d) culturing the infected grown MDCK-derivedcells under conditions allowing cloning of the influenza virus; and (e)isolating the influenza virus from the cell culture composition.
 14. Themethod according to claim 13, further comprising adding a fresh mediumto the cell culture or replacing a portion of the medium with a freshmedium in step (b).
 15. A virus or a virus antigen produced by themethod according to claim
 13. 16. A virus or a virus antigen produced bythe method according to claim
 14. 17. A method for producing aninfluenza virus from a cell culture, the method comprising: (a)inoculating a serum-free culture medium with the MDCK-derived cellaccording to claim 5 at a concentration of 1×10⁴ to 1×10⁶ cells/ml; (b)allowing the MDCK-derived cells to grow in a disposable bioreactorsystem until the cell density reaches at least 5×10⁶ cells/ml,comprising culturing the MDCK-derived cells while maintaining one ormore culture conditions selected from the group consisting of a stirringrate of 40 to 100 rpm, a pH of 6.5 to 7.5 and a dissolved oxygen (DO)concentration of 35 to 100%; (c) infecting the grown MDCK-derived cellswith an influenza virus; (d) culturing the infected grown MDCK-derivedcells under conditions allowing cloning of the influenza virus; and (e)isolating the influenza virus from the cell culture composition.
 18. Themethod according to claim 17, further comprising adding a fresh mediumto the cell culture or replacing a portion of the medium with a freshmedium in step (b).
 19. A virus or a virus antigen produced by themethod according to claim
 17. 20. A virus or a virus antigen produced bythe method according to claim
 18. 21. A method for producing aninfluenza virus from a cell culture, the method comprising: (a)inoculating a serum-free culture medium with the MDCK-derived cellaccording to claim 9 at a concentration of 1×10⁴ to 1×10⁶ cells/ml; (b)allowing the MDCK-derived cells to grow in a disposable bioreactorsystem until the cell density reaches at least 5×10⁶ cells/ml,comprising culturing the MDCK-derived cells while maintaining one ormore culture conditions selected from the group consisting of a stirringrate of 40 to 100 rpm, a pH of 6.5 to 7.5 and a dissolved oxygen (DO)concentration of 35 to 100%; (c) infecting the grown MDCK-derived cellswith an influenza virus; (d) culturing the infected grown MDCK-derivedcells under conditions allowing cloning of the influenza virus; and (e)isolating the influenza virus from the cell culture composition.
 22. Themethod according to claim 21, further comprising adding a fresh mediumto the cell culture or replacing a portion of the medium with a freshmedium in step (b).
 23. A virus or a virus antigen produced by themethod according to claim
 21. 24. A virus or a virus antigen produced bythe method according to claim
 22. 25. A method for preparing anMDCK-derived cell line that does not require serum for cell growth andis prepared by suspension culture without the need to be attached tocarriers, the method comprising: (a) preparing original MDCK cellsdeposited under accession number ATCC CCL-34; (b) adapting the originalMDCK cells to a serum-free medium to allow the original MDCK cells togrow in the serum-free medium; and (c) adapting the adherent MDCK cellsadapted in step (b) to a serum-free medium to allow the MDCK cells togrow in a suspension state without the need for carriers.